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Abstract

Background

The study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank
considerably during the last few years, resulting in the cessation of the production
of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein,
a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals.

Methods

Plasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study.
The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected
red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA
labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy.

Results

The results show that the sensitive strain has a higher Fluo-3/AM fluorescence in
a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM
fluorescence levels were negligible and increased only at high concentrations of Cd
and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from
cultures infected with either sensitive or resistant strain. In addition, confocal
microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates
in the parasite itself.

Conclusions

The dual fluorescent labelling is a valid method for detecting heavy metal accumulation
in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent
labelling enables us to differentiate between the different populations existing in
a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level
of a single cell.

Keywords:

Background

The development of the in vitro culturing technique of Plasmodium falciparum by Trager and Jensen
[1] revolutionized the understanding of various aspects in the life cycle of this deadly
parasite. The adoption of this technique in the research of malaria enabled the development
of biochemical, physiological, immunological and genetic techniques
[2-5] which resulted in a more comprehensive understanding of the biology of the parasite.
In spite of this advancement, falciparum malaria remains the most prevalent infectious
disease resulting in a high level of morbidity and mortality
[6]. The major reasons for this state is the lack of an appropriate vaccine
[7,8] and the evolving phenomenon of anti-malarial drug resistance
[9,10]. The gaps which still exist in understanding the biology of the parasite contribute,
although to a lesser extent, to this state. Since much of the studies on P. falciparum are done on the intact infected red blood cell (RBC) unit, it is preferable to be
able to localize unequivocally the activity within the infected unit. This paper describes
an assay of that nature which localizes heavy metal resistance to the parasite itself.
By comparing heavy metal resistant and sensitive P. falciparum lines, it was previously shown
[11] that this property, which prevents the accumulation of the heavy metal, is probably
determined by the parasite's P. falciparum multidrug resistance 2 (pfmdr2) gene. However, as the labelling technique used a radioactive heavy metal, it was
not possible to assess the resistance/sensitivity state at the level of a single infected
RBC whereas the technique described in this paper allows it. This was achieved by
dual labelling with Fluo-3/AM which labels the heavy metal whose transport is studied
and Hoechst which labels the parasite by binding to its DNA. Fluo-3/AM was scarcely
used in the study of P. falciparum and in those cases it served for determination of Ca+2 metabolism
[12-15]. Hoechst and derivatives is used more commonly and in all cases for the visualization
of the parasite e.g.
[16-20]. No dual labelling with these two dyes was reported hitherto in the literature.

Methods

Parasites and culture conditions

The isolate P. falciparum FCR3 and a cadmium resistant line originating from it were used throughout this study.
The in vitro culturing and synchronization of the parasites were carried out by standard protocols
[1,21]. Briefly, the parasites were cultured in flasks at 37°C and 5% haematocrit in RPMI
1640 medium supplemented with human heat inactivated (30min at 56°C) plasma (A+ or AB+), 50μg/ml gentamycin, 25mM HEPES, and 0.25% sodium bicarbonate in an atmosphere of
5% O2 ,5% CO2 and 90% N2. The wild-type FCR3 line demonstrates sensitivity to heavy metal exposure, whereas
the line originating from it by culturing it in the presence of a low concentration
of CdCl2 exhibits a resistant phenotype, up to a concentration of 500 nM CdCl2[11] . This ability is not cadmium-dependent, and this line is therefore cultured in its
absence
[11]. The resistant line was then used in the comparative studies with the wild-type line
unexposed to CdCl2.

Cd uptake and staining procedure

P. falciparum Cd resistant and sensitive strains infected RBCs at the trophozoite stage were cultured
in a U shaped 96-well plate (105 cells/well) in 200μl for indicated times (5-45min, or for 20min for concentration
dependent experiments) at 37°C with indicated CdCl2 concentrations (0.01-1μM, or 0.1μM for time dependent experiments), followed by three
washes. Fluo-3/AM (Invitrogen detection technologies) staining was at a concentration
of 5μM for 20min at 37°C, followed by two washes and incubation for 20min at 37°C.
Hoechst 33342 (Fluka) staining was performed for 30min on ice at a concentration of
20μM. After staining, the cells were held on ice until analysed by flow cytometry
and confocal microscopy. All procedures and washes were done in Dulbecco's Phosphate
Buffered Saline (DPBS) (without Ca2+ and Mg2+) containing 3.5% glucose. The addition of glucose enabled the maintenance of the
pfmdr2 ATP dependent transporter activity. Experiments were done at 8-12% parasitaemia,
where in each experiment parasitaemia for the sensitive and resistant strains were
the same.

Quantitation analysis

Flow cytometry

Stained infected RBCs were analysed by the fluorescence of Hoechst and Fluo-3/AM.
Hoechst fluorescence was excited using a violet laser at 405nm and measured with a
450/50nm filter. Fluo-3/AM fluorescence was excited using a blue laser at 488nm and
measured with a 530/30nm filter. Flow cytometry was performed using FACSCanto II (BD
Bioscience) and results were analysed using FlowJo software (Tree Star).

Confocal microscopy

Stained infected RBCs were applied to a chambered μ-slide (ibiTreat, #80826, ibidi).
The slides were mounted to the Olympus Fluoview FV1000 confocal microscope (Olympus:
UPLSAPO 60X Oil NA:1.35). Fields containing infected RBCs were selected and images
were acquired. Hoechst fluorescence was detected using a 405nm laser and measured
in the 461nm channel; Fluo-3/AM fluorescence was detected using a 488nm laser and
measured in the 527nm channel. Fields with infected RBCs were selected and images
were acquired with indicated zoom magnitudes.

Results and discussion

To evaluate the Cd uptake by the malaria parasite we used Cd labelled with the fluorescent
dye Fluo-3/AM. Flow cytometry was performed for quantitative evaluation and confocal
microscopy for qualitative measurement. Compared to the previous method of radioactive
Cd detection
[11], the fluorescent detection allows to differentiate between the fluorescence of the
infected RBC and that of the non-infected one. This separation is of utmost importance
as it allows the detection of low number of infected RBCs in the total cell population.
For detection of infected RBCs we used the DNA dye Hoechst 33342. As RBCs lack DNA,
the only population that will be stained will be that of the infected RBCs due to
the staining of the parasite's DNA. Hoechst staining to identify RBC infected with
malaria has been used in many studies e.g. [16–20].

Using this approach it can be seen that Hoechst staining clearly differentiates, by
flow cytometry, between two RBCs populations of high and low Hoechst fluorescence
(Figure
1A). Furthermore, analysis of these two populations using confocal microscopy shows
unequivocally that: a) the high intensity Hoechst population consists of only infected
RBCs, whereas the low intensity population contains only non-infected cells (Figure
1B, two upper panels). b) Only the parasite within the RBC is stained (Figure
1B, lower panels). Thus, the Hoechst staining enables us to focus directly on the infected
RBCs.

To assess the fluorescent method of Fluo-3/AM staining for Cd uptake, the P. falciparum pfmdr2 system, an ABC-type transporter that is involved in heavy metal homeostasis
[11] was used. In this system, the Cd-resistant strain has a functional form of pfmdr2 gene, enabling its survival in the presence of high Cd concentrations. On the other
hand, the sensitive strain has an impaired form of this gene, due to a premature translational
termination, thus it cannot survive the high concentrations of Cd accumulated
[11]. First, the uptake at different Cd concentrations was assessed. The results show
that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration
dependent manner, while in the resistant strain Fluo-3/AM fluorescence levels increases
only at high concentrations of Cd, but to a lesser extent than the sensitive line
(Figure
2A-B). Similar results were obtained by confocal microscopy (Figure
2C). Furthermore, the confocal microscopy overlay of Fluo-3/AM and Hoechst staining
(Figure
2C enlarged image) shows that the Fluo-3/AM and the Hoechst staining are localized
within the parasite (yellow arrow). No Cd uptake is observed in uninfected RBCs populations
originating from cultures infected either with the sensitive or the resistant lines
(Figure
2A-C). These last two points demonstrate that the Cd uptake measured by Fluo-3/AM is
specific to infected RBCs, and that this Cd uptake accumulates mainly within the parasite.

Figure 2.Concentration dependent accumulation of Cd in RBCs infected with P. falciparum Cd resistant and sensitive strains. (A-B) Fluo-3/AM fluorescence in Cd concentration dependent manner analysed by flow cytometry,
Sensitive and Resistant denote RBCs infected with sensitive and resistant parasite
respectively. (A) Two dimensional flow cytometry dot plots of stained populations of RBCs with Fluo-3/AM
and Hoechst. (B) After gating the populations for positive and negative to Hoechst, at a threshold
of 1.5*103 Hoechst fluorescent intensity, Fluo-3/AM fluorescence geometric mean was calculated.
The samples without Cd served as control. RBC-s and RBC-r denote non-infected RBCs
originating from the sensitive and resistant cultures respectively. Each time point
is a mean of duplicates, Bars ± SD. (C) Confocal images: I: Hoechst (Blue), II: Fluo-3/AM (Green), III: combined Hoechst
and Fluo-3/AM, IV: combined Hoechst and Fluo-3/AM with DIC (also shown in the enlarged
image). Yellow arrow points to Fluo-3/AM overlapping with Hoechst. [Red scale bar
= 20μm]. For the sensitive and resistant strains parasitaemia was 11%. The results
are from one representative experiment of three performed.

To further characterize the Cd accumulation by Fluo-3/AM in comparison to the radioactive
method, a time curve of Cd uptake experiments was performed. As measured by flow cytometry,
Fluo-3/AM fluorescence increases in a time dependent manner in the RBCs infected with
the P. falciparum sensitive line whereas the level of fluorescence in those infected with the resistant
line was low and not time dependent (Figure
3A). In accordance with the results in Figure
2, the uninfected RBCs did not show any change in fluorescence levels with incubation
time (Figure
3A). The confocal microscopy images confirmed the results obtained by flow cytometry
(Figure
3B), namely, levels of Fluo-3/AM increase in the sensitive strain in a time dependent
manner.

Figure 3.Time dependent Cd accumulation in RBCs infected with P. falciparum Cd resistant and sensitive strains. (A) Fluo-3/AM fluorescence in a time dependent manner analysed by flow cytometry. After
gating the populations for positive and negative to Hoechst, Fluo-3/AM fluorescence
geometric mean was calculated. The samples without Cd served as control. Sensitive
and resistant denote RBCs infected with sensitive and resistant parasite respectively.
RBC-s and RBC-r denote non-infected RBCs originating from the sensitive and resistant
cultures respectively. Each time point is a mean of duplicates, Bars ± SD. (B) Confocal images: I: Hoechst (Blue), II: Fluo-3/AM (Green), III: combined Hoechst
and Fluo-3/AM, IV: combined Hoechst and Fluo-3/AM with DIC. [Red scale bar = 20μm].
The results are from one representative experiment of two performed. For the sensitive
and resistant strains parasitaemia was 10%.

Conclusions

The results demonstrate that Fluo-3/AM fluorescent labelling is a reliable method
for following a physiological process occurring in living cells, in this case the
active accumulation of Cd in P. falciparum infected RBCs. The method is time and concentration dependent, the two basic biological
parameters needed for the characterization of a biological process. It is also shown
that the use of Hoechst dye enables the distinction between the different populations
existing in a P. falciparum infected RBCs culture and thus actually study a phenomenon at the level of a single
cell. This is the major advantage of this procedure over the previous radioactive
method which yielded information only on the entire population. The method described
here can be used both for quantitative measurements (flow cytometry) as well as for
qualitative assessments (confocal microscopy). As was shown recently for studying
oxidative stress in P. falciparum infected RBCs
[22], the basic idea underlying this method can be extended to the study of other properties
of the parasite once the appropriate fluorescent tag is available.

Abbreviations

Competing interests

The authors declare that they do not have any competing interests.

Authors’ contributions

BR designed, performed and analysed the study, wrote the manuscript. UH and RS conducted
experimental procedures in confocal analysis and culturing the parasites respectively.
AB participated in confocal microscopy design and analysis of experiments. AP was
involved in designing experiments and critical evaluation of the manuscript. YP conceptualized
the study, designed the experiments, analysed the results and wrote the manuscript.
All authors read and approved the final manuscript.

Acknowledgments

AB acknowledges the support of the Israel Science Foundation, grants: 1165/08 and
1661/08.